The present invention is concerned with the isolation of a gene of Salmonella which, when genetically disrupted, reduces both virulence and septic shock caused by this organism and increases sensitivity to agents which promote eradication of the bacteria, e.g., chelating agents. The nucleotide sequence of this gene and the means for its genetic disruption are provided, and examples of the use of tumor-targeted bacteria which possess a disruption in this gene to inhibit growth of cancers, including, but not limited to, melanoma, colon cancer, and other solid tumors are described.
Citation or identification of any reference in Section 2 of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
A major problem in the chemotherapy of solid tumor cancers is delivery of therapeutic agents, such as drugs, in sufficient concentrations to eradicate tumor cells while at the same time minimizing damage to normal cells. Thus, studies in many laboratories are directed toward the design of biological delivery systems, such as antibodies, cytokines, and viruses for targeted delivery of drugs, pro-drug converting enzymes, and/or genes into tumor cells. Houghton and Colt, 1993, New Perspectives in Cancer Diagnosis and Management 1: 65-70; de Palazzo, et al., 1992a, Cell. Immunol. 142:338-347; de Palazzo et al., 1992b, Cancer Res. 52: 5713-5719; Weiner, et al., 1993a, J. Immunotherapy 13:110-116; Weiner et al., 1993b, J. Immunol. 151:2877-2886; Adams et al., 1993, Cancer Res. 53:4026-4034; Fanger et al., 1990, FASEB J. 4:2846-2849; Fanger et al., 1991, Immunol. Today 12:51-54; Segal, et al., 1991, Ann N.Y. Acad. Sci. 636;288-294; Segal et al., 1992, Immunobiology 185:390-402; Wunderlich et al., 1992; Intl. J. Clin. Lab. Res. 22:17-20; George et al., 1994, J. Immunol. 152:1802-1811; Huston et al., 1993, Intl. Rev. Immunol. 10:195-217; Stafford et al., 1993, Cancer Res. 53:4026-4034; Haber et al., 1992, Ann. N.Y. Acad. Sci. 667:365-381; Haber, 1992, Ann. N.Y. Acad. Sci. 667: 365-381; Feloner and Rhodes, 1991, Nature 349:351-352; Sarver and Rossi, 1993, AIDS Research and Human Retroviruses 9:483-487; Levine and Friedmann, 1993, Am. J. Dis. Child 147:1167-1176; Friedmann, 1993, Mol. Genetic Med. 3:1-32; Gilboa and Smith, 1994, Trends in Genetics 10:139-144; Saito et al., 1994, Cancer Res. 54:3516-3520; Li et al., 1994, Blood 83:3403-3408; Vieweg et al., 1994, Cancer Res. 54:1760-1765; Lin et al., 1994, Science 265:666-669; Lu et al., 1994, Human Gene Therapy 5:203-208; Gansbacher et al., 1992, Blood 80:2817-2825; Gastl et al., 1992, Cancer Res. 52:6229-6236.
Regarding bacteria and cancer, an historical review reveals a number of clinical observations in which cancers were reported to regress in patients with bacterial infections. Nauts et al., 1953, Acta Medica. Scandinavica 145:1-102, (Suppl. 276) state:
The treatment of cancer by injections of bacterial products is based on the fact that for over two hundred years neoplasms have been observed to regress following acute infections, principally streptococcal. If these cases were not too far advanced and the infections were of sufficient severity or duration, the tumors completely disappeared and the patients remained free from recurrence.
Shear, 1950, J. A.M.A. 142:383-390 (Shear), observed that 75 percent of the spontaneous remissions in untreated leukemia in the Children""s Hospital in Boston occurred following an acute episode of bacterial infection. Shear questioned:
Are pathogenic and non-pathogenic organisms one of Nature""s controls of microscopic foci of malignant disease, and in making progress in the control of infectious diseases, are we removing one of Nature""s controls of cancer?
Subsequent evidence from a number of research laboratories indicated that at least some of the anti-cancer effects are mediated through stimulation of the host immune system, resulting in enhanced immuno-rejection of the cancer cells. For example, release of the lipopolysaccharide (LPS) endotoxin by gram-negative bacteria such as Salmonella triggers release of tumor necrosis factor, TNF, by cells of the host immune system, such as macrophages, Christ et al., 1995, Science 268:80-83. Elevated TNF levels in turn initiate a cascade of cytokine-mediated reactions which culminate in the death of tumor cells. In this regard, Carswell et al., 1975, Proc. Natl. Acad. Sci. USA 72:3666-3669, demonstrated that mice injected with bacillus Calmette-Guerin (BCG) have increased serum levels of TNF and that TNF-positive serum caused necrosis of the sarcoma Meth A and other transplanted tumors in mice. Further, Klimpel et al., 1990, J. Immunol. 145:711-717, showed that fibroblasts infected in vitro with Shigella or Salmonella had increased susceptibility to TNF.
As a result of such observations as described above, immunization of cancer patients with BCG injections is currently utilized in some cancer therapy protocols. See Sosnowski, 1994, Compr. Ther. 20:695-701; Barth and Morton, 1995, Cancer 75 (Suppl. 2):726-734; Friberg, 1993, Med. Oncol. Tumor. Pharmacother. 10:31-36 for reviews of BCG therapy.
Although the natural biospecificity and evolutionary adaptability of parasites has been recognized for some time and the use of their specialized systems as models for new therapeutic procedures has been suggested, there are few reports of, or proposals for, the actual use of parasites as vectors.
Lee et al., 1992, Proc. Natl. Acad. Sci. USA 89:1847-1851 (Lee et al.) and Jones et al., 1992, Infect. Immun. 60:2475-2480 (Jones et al.) isolated mutants of Salmonella typhimurium that were able to invade HEp-2 (human epidermoid carcinoma) cells in vitro in significantly greater numbers than the wild type strain. The xe2x80x9chyperinvasivexe2x80x9d mutants were isolated under conditions of aerobic growth of the bacteria that normally repress the ability of wild type strains to invade HEp-2 animal cells. However, Lee et al. and Jones et al. did not suggest the use of such mutants as therapeutic vectors, nor did they suggest the isolation of tumor-specific bacteria by selecting for mutants that show infection preference for melanoma or other cancers over normal cells of the body. Without tumor-specificity or other forms of attenuation, such hyperinvasive Salmonella typhimurium as described by Lee et al. and Jones et al. would likely be pan-invasive, causing wide-spread infection in the cancer patient.
Genetically engineered Salmonella have been demonstrated to be capable of tumor targeting, possess anti-tumor activity and are useful in delivering effector genes such as the herpes simplex thymidine kinase (HSV TK) to solid tumors (Pawelek et al., WO 96/40238). Two significant considerations for the in vivo use of bacteria are their virulence and ability to induce tumor necrosis factor xcex1 (TNFxcex1)-mediated septic shock. As TNFxcex1-mediated septic shock is among the primary concerns associated with bacteria, modifications which reduce this form of an immune response would be useful because TNFxcex1 levels would not become toxic, and a more effective concentration and/or duration of the therapeutic vector could be used.
Modifications to the lipid composition of tumor-targeted bacteria which alter the immune response as a result of decreased induction of TNFxcex1 production were suggested by Pawelek et al. (Pawelek et al., WO 96/40238). Pawelek et al. provided methods for isolation of genes from Rhodobacter responsible for monophosphoryl lipid A (MLA) production. MLA acts as an antagonist to septic shock. Pawelek et al. also suggested the use of genetic modifications in the lipid A biosynthetic pathway, including the mutation firA, which codes for the third enzyme UDP-3-O (R-30 hydroxylmyristoly)-glucosamine N-acyltransferase in lipid A biosynthesis (Kelley et al., 1993, J. Biol. Chem. 268: 19866-19874). Pawelek et al. showed that mutations in the firA gene induce lower levels of TNFxcex1. However, these authors did not suggest enzymes which modify the myristate portion of the lipid A molecule. Furthermore, Pawelek et al. did not suggest that modifications to the lipid content of bacteria would alter their sensitivity to certain agents, such as chelating agents.
In Escherichia coli, the gene msbB (mlt) which is responsible for the terminal myristalization of lipid A has been identified (Engel, et al., 1992 J. Bacteriol. 174:6394-6403; Karow and Georgopoulos 1992 J. Bacteriol. 174: 702-710; Somerville et al., 1996 J. Clin. Invest. 97: 359-365). Genetic disruption of this gene results in a stable non-conditional mutation which lowers TNFxcex1 induction (Somerville et al., 1996 J. Clin. Invest. 97: 359-365). These references, however, do not suggest that disruption of the msbB gene in tumor-targeted Salmonella vectors would result in bacteria which are less virulent and more sensitive to chelating agents.
The problems associated with the use of bacteria as gene delivery vectors center on the general ability of bacteria to directly kill normal mammalian cells as well as their ability to overstimulate the immune system via TNFxcex1 which can have toxic consequences for the host (Bone, 1992 JAMA 268: 3452-3455; Dinarello et al., 1993 JAMA 269: 1829-1835). In addition to these factors, resistance to antibiotics can severely complicate coping with the presence of bacteria within the human body (Tschape, 1996 D T W Dtsch Tierarztl Wochenschr 1996 103:273-7; Ramos et al., 1996 Enferm Infec. Microbiol. Clin. 14: 345-51).
Hone and Powell, WO97/18837 (xe2x80x9cHone and Powellxe2x80x9d), disclose methods to produce gram-negative bacteria having non-pyrogenic Lipid A or LPS. Although Hone and Powell broadly asserts that conditional mutations in a large number of genes including msbB, kdsA, kdsB, kdtA, and htrB, etc. can be introduced into a broad variety of gram-negative bacteria including E. coli, Shigella sp., Salmonella sp., etc., the only mutation exemplified is an htrB mutation introduced into E. coli. Further, although Hone and Powell propose the therapeutic use of non-pyrogenic Salmonella with a mutation in the msbB gene, there is no enabling description of how to accomplish such use. Moreover, Hone and Powell propose using non-pyrogenic bacteria only for vaccine purposes.
The objective of a vaccine vector is significantly different from the presently claimed tumor-targeted vectors. Thus, vaccine vectors have requirements quite different from tumor-targeted vectors. Vaccine vectors are intended to elicit an immune response. A preferred live bacterial vaccine must be immunogenic so that it elicits protective immunity; however, the vaccine must not be capable of excessive growth in vivo which might result in adverse reactions. According to the teachings of Hone and Powell, a suitable bacterial vaccine vector is temperature sensitive having minimal replicative ability at normal physiological ranges of body temperature.
In contrast, preferred tumor-targeted parasitic vectors, such as but not limited to Salmonella, are safely tolerated by the normal tissues of the body such that pathogenesis is limited, yet the vectors target to tumors and freely replicate within them. Thus, vaccine vectors which replicate minimally at normal body temperatures, would not be suitable for use as tumor-targeted vectors.
The present invention provides a means to enhance the safety of tumor-targeted bacteria, for example, by genetic modification of the lipid A molecule. The modified tumor-targeted bacteria of the present invention induce TNFxcex1 less than the wild type bacteria and have reduced ability to directly kill normal mammalian cells or cause systemic disease compared to the wild type strain. The modified tumor-targeted bacteria of the present invention have increased therapeutic efficacy, i.e., more effective dosages of bacteria can be used and for extended time periods due to the lower toxicity in the form of less induced TNFxcex1 and systemic disease.
The present invention provides compositions and methods for the genetic disruption of the msbB gene in bacteria, such as Salmonella, which results in bacteria, such as Salmonella, possessing a lesser ability to elicit TNFxcex1 and reduced virulence compared to the wild type. Additionally, the genetically modified bacteria have increased sensitivity to a chelating agent compared to bacteria with the wild type msbB gene. In a preferred embodiment, Salmonella, which are hyperinvasive to tumor tissues, are able to replicate within the tumors, and are useful for inhibiting the growth and/or reducing the tumor volume of sarcomas, carcinomas, lymphomas or other solid tumor cancers, such as germ line tumors and tumors of the central nervous system, including, but not limited to, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma.
In an embodiment of the present invention, the bacteria are attenuated by other means, including but not limited to auxotrophic mutations. In another embodiment, the bacteria express pro-drug converting enzymes including but not limited to HSV-TK, cytosine deaminase (CD), and p450 oxidoreductase.
The present invention also provides a means for enhanced sensitivity for use in terminating therapy and for post therapy elimination. According to one embodiment of the present invention, the tumor-targeted bacteria having a genetically modified lipid A also have enhanced susceptibility to certain agents, e.g., chelating agents. It is a further advantage to modify tumor-targeted bacteria in this way because it increases the ability to eliminate the bacteria with agents which have an antibiotic-like effect, such as chelating agents including, but not limited to, Ethylenediaminetetraacetic Acid (EDTA), Ethylene Glycol-bis(xcex2-aminoethyl Ether) N, N, Nxe2x80x2, Nxe2x80x2,-Tetraacetic Acid (EGTA), and sodium citrate. Modification to enhance the ability to eliminate the bacteria via exogenous means, such as the administration of an agent to which the genetically modified bacteria are more sensitive than their wild type counterparts, is therefore useful.
As used herein, Salmonella encompasses all Salmonella species, including: Salmonella typhi, Salmonella choleraesuis, and Salmonella enteritidis. serotypes of Salmonella are also encompassed herein, for example, typhimurium, a subgroup of Salmonella enteritidis, commonly referred to as Salmonella typhimurium. 
Attenuation: Attenuation is a modification so that a microorganism or vector is less pathogenic. The end result of attenuation is that the risk of toxicity as well as other side-effects is decreased, when the microorganism or vector is administered to the patient.
Virulence: Virulence is a relative term describing the general ability to cause disease, including the ability to kill normal cells or the ability to elicit septic shock (see specific definition below).
Septic shock: Septic shock is a state of internal organ failure due to a complex cytokine cascade, initiated by TNFxcex1. The relative ability of a microorganism or vector to elicit TNFxcex1 is used as one measure to indicate its relative ability to induce septic shock.
Chelating agent sensitivity: Chelating agent sensitivity is defined as the effective concentration at which bacteria proliferation is affected, or the concentration at which the viability of bacteria, as determined by recoverable colony forming units (c.f.u.), is reduced.
The present invention may be understood more fully by reference to the following detailed description, illustrative examples of specific embodiments and the appended figures.
FIGS. 1A-B. The complete DNA sequence of the Salmonella wild-type (WT) 14028 msbB gene (SEQ ID NO:1) and the deed amino acid sequence of the encoded protein (SEQ ID NO:2).
FIGS. 2A-2C. Knockout construct generated using the cloned Salmonella WT 14028 msbB gene. The cloned gene was cut with SphI and MluI thereby removing approximately half of the msbB coding sequence, and the tetracycline resistance gene (TET) from pBR322 cut with AatII and Aval was inserted after blunting using the Klenow fragment of DNA polymerase I. A=Knockout construct B=Salmonella chromosomal copy of msbB. C=Salmonella disrupted chromosomal copy of msbB after homologous recombination. The start codon (ATG) and stop codon (TAA) and restriction sites Asel, BamHI, SphI, Mlul, and EcoRV are shown. The position of two primers, P1 and P2 which generate two different sized PCR products for either wild type or disrupted msbB are shown.
FIGS. 3A-3C. Southern blot analysis of chromosomally disrupted Salmonella WT 14028 msbB. A) Southern blot probed with the tetracycline gene, demonstrating its presence in the plasmid construct and the two clones, and its absence in the WT 14028bacteria B) Southern blot of a similar gel probed with an 32P-labeled Asel/BamHl fragment derived from the cloned msbB. The Asel enzyme cuts upstream of msbB, and the BamHl cuts in one location in the wild type, but in a second location in the tetracycline gene which results in a higher molecular weight product Lane 1 (KO) shows the position of the band in the knockout construct, compared to the WT 14028 in lane 2 (WT). Lanes 3 and 4 show the clones YS8211 and YS861 with a higher molecular weight product. C) Southern blot of a similar gel probed with an 32P-labeled mluI fragment derived from the cloned msbB. See text Section 7.2 for details.
FIG. 4. TNFxcex1 induction by live Salmonella WT 14028 in mice. 1xc3x97108 live bacteria in 0.1 cc phosphate buffered saline of the wild type or msbBxe2x88x92 disrupted strains were injected i.v. in the tail vein of Balb/c mice. The bar graph indicates the TNFxcex1 induction with error bars. Clone YS8211 induces TNFxcex1 32% compared to Salmonella WT 14028.
FIG. 5. TNFxcex1 response by Sinclair swine to live Salmonella WT 14028 and msbBxe2x88x92 clone YS8212. TNFxcex1 levels were measured at 1.5 and 6.0 hours following i.v. introduction of 1xc3x97109 c.f.u. Salmonella WT 14028 and YS8212. At 1.5 hours TNFxcex1 response was significantly lower (pxe2x89xa60.011) in the msbB deletion mutant compared to the wild type.
FIGS. 6A-6B. Respiratory level changes induced by LPS from WT 14028 and msbBxe2x88x92 clone YS8212. Sinclair swine were injected with 5 or 500 xcexcg/kg purified LPS and respiration rate was determined. The 500 xcexcg/kg of LPS from Salmonella WT 14028 raised the rate of respiration to more than 4 times normal, whereas the rate of respiration in msbBxe2x88x92LPS-treated animals, was less than doubled.
FIG. 7. TNFxcex1 induction by live Salmonella WT 14028 in human monocytes. Human monocytes isolated from peripheral blood were exposed to increasing amounts of Salmonella c.f.u. At 1.0xc3x97105 c.f.u., concentrations of TNFxcex1 induced by WT 14028 were more than 3 times higher than those induced by a number of msbBxe2x88x92 clones, i.e., YS8211, YS8212, YS8658, and YS1170.
FIG. 8. TNFxcex1 production by human monocytes. Human monocytes isolated from peripheral blood were exposed to increasing amounts of purified LPS. As little as 1 nanogram of LPS from wild type was sufficient to elicit a measurable TNFxcex1 response and was maximal at 10 ng. In contrast, 100 xcexcg of LPS from each of a number of msbBxe2x88x92 clones was insufficient to generate any response. Thus, at 10 ng LPS, the concentration of TNFxcex1 induced by Salmonella WT 14028 was at least 105 times higher than concentrations of TNFxcex1 induced by the independent msbB knockouts, i.e., YS7216 and YS8211, and the derivatives, i.e., YS1170, YS8644, YS1604, YS8212, YS8658, YS1601, YS1629.
FIGS. 9A-9B. Survival of mice and Sinclair swine, injected with 2xc3x97107 or 1xc3x97109 respectively of live bacteria A) WT 14028 killed all the mice in 4 days, whereas the msbBxe2x88x92 clone YS862 spared 90% of the mice past 20 days. B) Similarly, WT 14028 killed all the swine in 3 days, whereas the msbBxe2x88x92 clone YS8212 spared 100% of the swine past 20 days.
FIG. 10. Biodistribution of msbBxe2x88x92 Salmonella YS8211 in B16F10 melanoma tumors. At 5 days, the ratio of msbBxe2x88x92 Salmonella within the tumors compared to those in the liver exceeded 1000:1.
FIG. 11. Tumor retardation by msbBxe2x88x92 Salmonella. B16F10 melanoma tumors were implanted in the flank of C57BL/6 mice and allowed to progress to day 8. Mice either received no bacteria (control) or msbBxe2x88x92 strains YS8211, YS8212, YS7216, YS1629. Two of the strains, YS8211 and YS1629 retarded tumor progression significantly, whereas strains YS7216 and YS8212 did not.
FIGS. 12A-12B. Sensitivity of WT 14028 and msbB disrupted bacteria to chelating agents. Wild type and msbB disrupted Salmonella clone YS8211 and YS862 were grown in LB broth lacking sodium chloride (LB-zero), in the presence or absence of 1 mM EDTA (FIG. 12A) or in the presence or absence of 10 mM sodium citrate (FIG. 12B). The OD600 was determined and plotted as a function of time. The msbB+ strain showed little inhibition by EDTA or sodium citrate, compared to the msbBxe2x88x92 strains which showed near complete cessation of growth after 3 hours for EDTA sodium citrate.
FIGS. 13A-13B. Survival of msbBxe2x88x92 bacteria within murine macrophages. Murine bone marrow-derived macrophages (FIG. 13A) and a murine macrophage cell line, J774, (FIG. 13B) were used as hosts for bacterial internalization and quantified over time. The data are presented as a percentage of initial c.f.u.